Project Summary/Abstract Protein Phosphatase 2A (PP2A) is a ubiquitously expressed serine-threonine phosphatase. It has a direct role in multiple areas of cellular function by counter-balancing kinase-mediated phosphorylation. PP2A is a hetero- trimeric enzyme composed of a scaffolding A-subunit, a catalytic C-subunit, and one of several identified regulatory B-subunits. Study of PP2A inactivation in cancer has described its role as a tumor suppressor. One major mechanism through which PP2A is altered is somatic mutation to the gene encoding the A? scaffolding subunit isoform. Notably, A? mutations include several hotspot sites that were recurrently mutated across multiple independent patient tumors. These sites clustered to a specific structural region of the protein, where the A-subunit makes contact with regulatory B-subunits. Moreover, two mutation sites, residues P179 and S256, were highly mutated but almost exclusively in endometrial carcinomas (EMCA). 27.3% of serous-type endometrial carcinomas and 26.8% of endometrial carcinosarcomas harbored A? mutations, and represented the cancers with the highest A? mutation rate overall by more than three-fold. Preliminary characterization of mutant proteins revealed shifts in A? conformational dynamics and altered binding with PP2A B- and C- subunits. Mutant proteins are presumed to have altered regulatory activity against PP2A substrates due to their impairment of canonical holoenzyme assembly. However, their impact on PP2A-regulated signaling pathways has not been broadly characterized. Likewise, a role for PP2A-A? mutations as drivers of disease has not been directly assessed. A lack of well-established disease drivers for EMCA has greatly limited the pursuit of effective targeted therapeutic strategies. And while cancer-related deaths have been declining in the US, mortality rates for uterine cancers, of which EMCA accounts for the vast majority, have steadily increased. We therefore aim to better characterize hotspot P179 and S256 A? mutations that re-occur in EMCA, in order to enhance understanding of the pathologic mechanisms that facilitate this disease. The first aim of this project will directly interrogate cancer cell dependence on A? mutation as a pathogenic driver mutation, and determine its association with metastatic and invasive phenotypes. The second aim will consider deregulation of GSK3?- Axin signaling downstream of mutant PP2A-A?. Highlighting specific pathways with signaling perturbation can inform the rational design of new, targeted therapy.